The term “fusion protein” refers to a protein, obtained by an expression of a recombinant DNA molecule, in which coding arrays of several different genes are bound to one reading frame.
Annually, influenza virus affects from 5 to 15 percent of the population, causing acute respiratory diseases. Every year, vaccines against seasonal influenza are produced and widely used. However, a danger of pandemic spread of certain influenza virus strains with a new combination of genes exists. Such viruses can be more aggressive and cause a higher level of complications and fatalities. In 2009, an unfavorable situation took place, which was connected with an extension of the highly pathogenic influenza virus A(H1N1)pdm/09. Diseases were caused by the influenza virus with new antigenic determinants. In particular, specialists were worried about the fact that the new virus was a reassortant of viruses, circulating among both animals (swine) and humans. According to WHO, the infection ratio for humans, who had contact with contracted people, was 22% to 33% for A(H1N1) pdm/09. In comparison, the analog ratio for a seasonal influenza ranges from 5% to 15%.
The viral surface protein haemagglutinin (HA) is of particular interest in the light of development of the vaccines. HA is responsible for virus binding to cell receptors and in such a way for susceptibility to the disease. Mutations of HA usually lead to the increase of epidemiological and sometimes pandemic viral potential.
B-cell and T-cell response is being developed against antigenic determinants of viral proteins—the epitopes. Immunization against viral infections, namely influenza virus, based on usage of amino acid sequences represented by the viral epitopes, show great promise for the development of immune response against the virus and allows to create a universal vaccine for the prevention of both seasonal infection episodes and pandemic. Usage of antigenic determinants of different viral subtypes in one construct makes the production of the polyvalent vaccine easier and allows to create effective vaccines against new viral strains.
It is essential to create a universal vaccine, which would be able to provide defense against the existing and possible reassortant influenza virus strains. The usage of vaccines, based on recombinant proteins, allows to avoid risks, which are connected even with the injection of inactivated viruses. A number of attempts has been made recently to create such a universal safe vaccine.
There is an invention, according to which the gene, coding for a multiepitopic haemagglutinin of the avian influenza virus, is used within the vector construct for the production of the recombinant multiepitopic vaccine in plants (RU 2008139004 A, Oct. 1, 2008). In the patent applications US 2009106864 (2009) and RU 2008139004 the authors used the consensus sequence of the avian influenza A virus haemagglutinin, codon optimized for the expression in plants. In the patent application JP2009102416 (2009) the authors used baculovirus constructs in the insect cells for the expression of haemagglutinin of influenza A and B viruses. The disadvantage of such approaches is the usage of protein (haemagglutinin) sequence from only one influenza virus subtype (H5N1). This narrows the spectrum of the vaccine activity. Moreover, the expression in plants and insect cells provides lower protein yields in comparison to the expression in E. coli. 
Several fusion polypeptides are known which include a sequence with a high percentage of homology with the superficial HA-protein of the influenza A virus, containing five immunodominant antigenic epitopes (RU 92004487, Dec. 10, 1992). One of the suggested applications for such polypeptides is to provide vaccines against corresponding antigens, for treatment and prophylaxis (including immunotherapeutic methods) in case of infection by such antigens. However, it is not specified, haemagglutinin of which subtypes is used. In the course of the development of the universal vaccine, the antigenic determinants of haemagglutinin of the influenza B virus as well should be taken into consideration, because the immunogenicity of the vaccine is limited by the strains, the sequences of which are used.
A polyvalent influenza vaccine is known which contains a polyvalent anti-influenza antigen, an adjuvant, an agent that provides the vaccine penetration, and an acceptable excipient (CN101450209, Dec. 31, 2008). Antigens represented by H1-H16 and N1-N9 are declared as an antigenic component. A polyvalent influenza vaccine is also known consisting of surface viral proteins A/H1N1, A/H3N2, haemagglutinin of the B-type (CN101524538, Mar. 26, 2009), an adjuvant—glycerol or aluminum hydroxide. The usage of both an acceptable adjuvant and antigenic determinants in the fusion construct, as well as using single protein type—haemagglutinin will allow to simplify and reduce the cost for the production of the vaccine, inasmuch as it will be no more necessary to adjust the conditions and to control the quality of the component production.
A polyvalent vaccine is known which is based on the inactivated viruses. The active component is represented by antigens A/H1N1, H3N2, B, H5N1, and A (H1N1)pdm/09 (CN101732711, Dec. 31, 2009). The listed antigens are extracted from the inactivated viruses, which means that the scaled-up production of the vaccine can be limited by possible problems, connected with the usage of viruses, especially influenza viruses. For instance, it is necessary to create appropriate working conditions, to handle easily transmitted highly pathogenic virus strains. Moreover, the listed antigens are separate proteins—the advantages of usage of a fusion construct are listed above.
The prototype of the invention is a mixture of flagella, containing at least four protein epitopes of influenza virus, which interact with human cells, whereat each one is being expressed separately in the flagellin of Salmonella (WO0032228, Nov. 30, 1998). The listed epitopes refer to the group consisting of: (i) one haemagglutinin epitope, interacting with B cells, (ii) one epitope of haemagglutinin (HA) or of a nucleoprotein (NP), which can bind to MHC molecules for representation to receptors on the surface of the T helper cells and (iii) at least two nucleoprotein (NP) epitopes or a matrix protein (M) which are restricted by the MHC antigens that dominate by the Caucasians and bind to cytotoxic T lymphocytes (CTL).
In the present invention, flagellin FliC of Salmonella typhimurium fulfills an adjuvant function. Due to its interaction with Toll-like receptor-5 (TLR-5), FliC stimulates maturation of macrophages and dendritic cells, which results in induction of the immune response (Mc Dermott P. F. High-affinity interaction between Gram-negative flagellin and a cell surface polypeptide results in human monocyte activation. Infect. Immun.—2000.—V. 68.—p.: 5525-5529; Means T. K. et al. The Toll-like receptor 5 stimulus bacterial flagellin induces maturation and chemokine production in human dendritic cells. J. Immunol.—2003.—V. 170.—p.: 5165-5175).
At the moment, flagellin is one of the most promising and well-studied new-generation adjuvants. The results of studies show that recombinant proteins, inoculated together with flagellin, possess higher immunogenic and antigenic properties. The responses to them are registered in shorter times and induce a more intense cellular and humoral immune response (Balaram, 2008).
The disadvantage of such invention lies in the complexity of the immunizing mixture. The usage of a fused protein, containing B and T cell epitopes of different influenza A and B virus subtypes, and flagellin, will trigger an intense immune response and lower the production costs. Therewithal, it is practical to use only components of flagellin. Two receptor-activating sites were detected in the terminal sites of flagellin (aa 79-117 and aa 08-439) (Tonyia, 2001).
TLR-5 is expressed on the cells of the innate immunity, epithelial, and endothelial cells (Sebastiani G. et al. Cloning and characterization of the murine Toll-like receptor 5 (Tlr5) gene: sequence and mRNA expression studies in Salmonella-susceptible MOLF/Eimice. Genomics.—2000. —V. 64.—p. 230-240; Zarember K. A. and Godowski P. J. Tissue expression of human Toll-like receptors and differential regulation of Toll-like receptor mRNAs in leukocytes in response to microbes, their products, and cytokines. J. Immunol.—2002.—V. 168.—p. 554-561; Delneste, 2007). Taking this into consideration, it is practical to use mucosa for immunization, which will make the transport of the immunogen easier.